The present invention relates to a new mutant Rhizobium strain that is deficient in a gene involved in autoinducer synthesis. The invention also relates to a method of increasing the nitrogen fixation in Leguminosae and a method of providing a Rhizobium strain having a mutation in a gene involved in autoinducer synthesis and the product of this method.
Autoinduction is a highly conserved mechanism of differential gene expression in many Gram-negative bacteria. The key trigger of this system is the concentration of small diffusible molecules, termed autoinducers in respect to their biological activity. All autoinducers so far identified are N-acyl homoserine lactones (AHLs). They are synthesized by an autoinducer synthase, the product of a luxI-homologous gene, and are thought to bind to a protein belonging to the LuxR-family of transcriptional activators. This autoinducer-protein complex activates the expression of defined genes or sets of genes. As this gene activation occurs only when a required threshold concentration of AHLs is attained, the onset of specific genes is dependent on the cell density of bacteria. Consequently, autoinduction allows bacteria to monitor their own population density and to discriminate between high and low cell density. It can also be understood as a cell-cell communication system.
The physiological processes regulated by autoinduction are diverse, as exemplified by the following systems: bioluminescence in Vibrio fischeri, plasmid conjugal transfer in Agrobacterium tumefaciens, antibiotic production in Erwinia carotovora, and synthesis of exoenzymes in plant and animal pathogens like E. carotovora and Pseudomonas aeruqinosa. In Escherichia coli, a LuxR-homologue is involved in the regulation of cell division.
Rhizobium etli CNPAF512 (formerly classified as R. leguminosarum bv. Phaseoli CNPAF512) forms nitrogen-fixing nodules on the roots of common bean. Within this structure bacteria are densely packed and differentiate into their symbiotic state, the bacteroids, able to reduce atmospheric dinitrogen into ammonia. In view of the high cell density during bacteria-plant interaction, autoinduction may be involved at some stage of symbiosis.
Since the nitrogen-fixation that occurs in the nodules is important for plants, it is always desirable to be able to increase the nitrogen-fixing capacity per plant.
In the research that led to the present invention a Rhizobium etli CNPAF512 gene has now been identified by which the Rhizobium strain containing the gene is able to control the number of nodules formed per plant. Based on this information a mutant could be developed in which this particular gene is inactivated and inactivation of this gene led to a significant increase in the number of nodules formed per plant.
The invention thus relates to a new mutant Rhizobium etli CNPAF512 strain, in which the biological function of the raiI gene is inactivated. More in particular the invention relates to a mutant Rhizobium etli CNPAF512 strain having a gusA-Km insertion in its raiI gene. More in particular, the invention provides a mutant Rhizobium etli CNPAF512 strain, herein designated as FAJ1328 and having a gusA-Km insertion in the XhoI restriction site of the raiI gene. The nucleotide sequence of the raiI gene is given in FIG. 1.
Based on the identification of the raiI gene in Rhizobium etli CNPAF512 it became possible according to the invention to create other mutant rhizobia strains by:
a) identifying a gene that is at least 40% similar with the raiI gene of Rhizobium etli CNPAF512 strain in any rhizobia strain; and
b) inactivating the biological function of the gene similar to raiI. In this application the term xe2x80x98similarxe2x80x99 intends to encompass both situations in which sequence homology exists and situations wherein a similar biological function (autoinduction) exists
Inactivation of the biological function can be achieved in various ways.
First, the gene similar to raiI can be mutated by either an insertion of any DNA sequence that is capable of disrupting the reading frame or by deletion of a part of the coding sequence, for example also leading to disruption of the reading frame. Mutations that lead to an internal stop codon can also be used to inactivate the biological function.
Second, the biological function of the raiI gene or other genes encoding autoinducer synthases can be inactivated by interfering with their transcription regulation factors. This interference leads to a complete or partial lack of transcription of the raiI gene. xe2x80x9cBiological functionxe2x80x9d is to be understood as comprising both the direct biological function of the gene, i.e. synthesis of the gene product encoded by the gene and the effect thereof, or its indirect biological function, e.g. synthesis of other molecules like autoinducers, the synthesis of which is dependent on the expression of the raiI gene, and the effect of the synthesis of those molecules. For example, for Rhizobium etli CNPAF512, one of the ultimate biological functions of the non-mutated raiI gene is limitation of the number of nodules formed upon infection of a species of the Leguminosae.
The rhizobia component of the nodules of Leguminosae is specific at least to the genus of the Leguminosae family members. A skilled person will very well be capable of selecting a suitable rhizobia species for the plant in which the nodule number should be increased. Starting from this rhizobia species mutants can be created in which the biological function of the raiI (homologous) gene or other genes encoding autoinducer synthases is inactivated. The rhizobia encompass inter alia the genus Rhizobium and the genus Bradyrhizobium.
The invention further relates to rhizobia strains being deficient in the biological function of their raiI gene or raiI homologous gene or other genes encoding an autoinducer synthases, obtainable by the method as described above.
According to a further aspect thereof, the invention relates to such mutant rhizobia strains for use in increasing the number of nodules in their corresponding host plants and to a method for increasing the nitrogen fixation in Leguminosae, comprising inoculating Leguminosae plants with a mutant strain and providing circumstances suitable for the rhizobia strain to induce nodules.
In a first preferred embodiment of the invention the Leguminosae plant is a bean plant and the rhizobia strain is a strain modulating bean such as Rhizobium leguminosarum biovar phaseoli.
In a second preferred embodiment the Leguminosae plant is a Phaseolus plant and the rhizobia strain is Rhizobium etli CNPAF512.
In a third preferred embodiment the Leguminosae plant is a Glycine max plant and the rhizobia strain is soy-bean nodulating strain selected from Bradyrhizobium japonicum CB1809 and Bradyrhizobium elkani BR29W.
According to still a further aspect of the invention there is provided the raiI gene of Rhizobium etli CNPAF512 in substantially isolated form, comprising a coding nucleotide sequence that is identical to the coding sequence of the nucleotide sequence depicted in FIG. 1 or at least 90% similar to this sequence, or the complementary sequence of either of these. This raiI gene can be used as a probe for screening other rhizobia strains for the presence of a gene that is at least 40% similar to raiI of Rhizobium etli CNPAF512.
The present invention will be further illustrated by the following examples. First it is demonstrated how the presence of the raiI gene was detected through its biological function. Then the relevant genes are isolated (Example 2). Creation of the mutant is described in Example 3, whereas Example 4 illustrates the effect of the mutation on the biological function. Example shows the detection of raiI similar genes in Bradyrhizobium. Example 6 shows mutagenesis of genes encoding autoinducer synthase in other rhizobia species. Example 7 shows the use of raiI as a probe for screening other rhizobia strains.